Science - USA (2020-06-05)

Nelidovaet al.,Science 368 , 1108–1113 (2020) 5 June 2020 2of6

Fig. 1. NIR light responses in mouse cone
photoreceptors.(A) Components of
the NIR light sensor. Engineered TRP
channels (blue) express protein epitope tags
(orange) in extracellular domains and
bind antibody-conjugated gold nanorods.
(B) Left: Healthy retina. Photons are
captured by outer segments (OS)
of photoreceptor cells. Right: retinal
degeneration, characterized by loss of OS
and blindness. In the rd1 mouse model
of degeneration, rod cell bodies are lost but
cone cell bodies persist. ONL, outer
nuclear layer; INL, inner nuclear layer; GCL,
ganglion cell layer. (CandD) Structure
of rTRPV1 channel. (C) Top view. Orange
arrows indicate a 6x-His epitope tag in each
of the four subunits of the TRPV1 tetramer
(yellow, blue, gray, and green). The red
asterisk marks the channel pore. (D) Right:
Side view. Left: 6x-His epitope tag (orange)
within the first extracellular loop of the
blue subunit is enlarged. (E) Top row:
Top views of rd1 retinas transduced with
both rTRPV1 and nanorods and immuno-
stained for TRPV1 (left, blue), 6x-His
(middle, orange), and merging the two
(right). Gray, Hoechst nuclear stain. Bottom
row: Cross sections of the retinas shown
in the top row. Scale bars, 25mm.
(F) Number of rTRPV1-positive (blue) and
6x-His–positive (orange) cones per square
millimeter in rd1 retinas transduced with both
rTRPV1 and nanorods (n=5mice)orin
control, uninjected rd1 retinas (n= 5 mice).
Dashed arrow indicates maximum cone
density in rd1 mice at postnatal day 70 ( 10 ).
Each data point is collected from a
different region of a retina (three regions per
retina). (G) Example calcium responses
(meanDF/F,whereFrepresents baseline
fluorescence; two to three repetitions)
recorded from cone axon terminals in
P56-P71 rd1 mice transduced with rTRPV1
and nanorods (left,labsnanorod = 915 nm),
in rd1 mice transduced with rTRPV1 only
(middle), or in wild-type mice (right).
TRP,n= 4 mice; wild-type,n= 3 mice.
Stimulus, full-field NIR light (915 nm, log 10
light intensity = 18.9; left and middle) or
visible light (405 nm, log 10 light intensity =
14; right). Black bars (2 s) and arrows
(100 ms) indicate stimulus timing. Two-
photon images of GCaMP6s-expressing cone
axon terminals (white circles) are shown
to the left of the response curves. Scale bar,
5 mm. White asterisks indicate cell bodies.
(H) Cumulative frequency of responding
rTRPV1-transduced rd1 cones with (black)
and without (gray) nanorods (labs= 915 nm).
(I) Cone response amplitudes (DF/F).
Light intensities as in (G). Number of cones
given as a ratio of responding cones to
measured cones.

C

rTRPV1 6x-His Merged

ONL

INL

GCL

ONL

rTRPV1

6x-His

Number of cones per mm

2

rTRPV1-nanorodsn = 5 retinas

Control

n = 5 retinas

Rd1. rTRPV1-nanorods

915 nm

Cones. GCaMP6s

405 nm

G

F

A

Rd1 mouse retina

Rd1. rTRPV1 only Wild-type

915 nm

(^12)
3

• (^12) 5 s
  3


• 
  

  1
  2
  3
  ONL
  INL
  GCL
  OS
  H
  B
  E
  Control
  rTRPV1-nanorodsn = 5 retinasn = 5 retinas
  Gold nanorod
  Antibody
  TRP channel
  17 18 19
  Cone photoreceptors

  
  


• 
  

  100 Å
  Top view Side views
  Extracellular loop 1
  D
  n.s.
  I
  rTRPV1-nanorods
  n = 57/152 cones
  rTRPV1 only
  n = 0/67 cones
  1
  0
  200
  400
  600
  800
  C
  A
  Gold nanorod
  Antibody
  TRP channel
  Wild type (WT) retina Rd1 (Pde6brd1) retina
  TRPV1.465-6x-His (rTRPV1)
  300%
  ΔF/F
  rTRPV1-nanorods
  n = 152 cones
  rTRPV1 only
  n = 67 cones
  Cumulative frequency of res
  ponding c
  ones (%)
  Log 10 I 915 (photons cm-2 s-1)
  Cone response amplitude (%
  ΔF/F)
  0
  4000
  8000
  12,000
  0
  10
  20
  30
  40
  RESEARCH | REPORT